Aromatic hydrocarbons (AH) are widespread pollutants within the environment owing to natural, industrial and social activities. Many AH are toxic, carcinogenic and recalcitrant within the environment, making their removal extremely important for public health. Biodegradation by microorganisms, especially bacteria, is considered the most cost-effective and appropriate way to remove AH from contaminated soils. While a huge body of literature exists for the intracellular fate of AH and other xenobiotics, virtually nothing is known about the uptake of such molecules. In Gram-negative bacteria, the outer membrane (OM) forms a very effective barrier against the spontaneous permeation of hydrophobic molecules, due to the presence of lipopolysaccharide (LPS) on the outside of the cell. As a consequence, OM protein channels are required to mediate passage of hydrophobic molecules into the cell. The goal of this project is to understand how AH are transported across the outer membrane (OM) of Gram-negative bacteria and to assess the importance of those channels for biodegradation under conditions resembling the natural environment. In addition we will perform genome-wide analyses to identify other proteins that are important during biodegradation of AH. The FadL family: OM channels for uptake of hydrophobic molecules. To date, the only OM proteins with an established role in the transport of hydrophobic molecules belong to the FadL family, members of which are widespread in biodegrading Gram-negative bacteria, including Pseudomonas putida F1 (PpF1). The archetype of the family, FadL from Escherichia coli (EcFadL), mediates uptake of long-chain fatty acids (LCFAs) across the OM. We have recently discovered that EcFadL-mediated LCFA uptake across the OM occurs via a unique, lateral diffusion mechanism. In addition, we have shown that EcFadL functions as a ligand-gated channel. Besides our work on EcFadL, we have determined crystal structures of FadL channels involved in mono-aromatic hydrocarbon (MAH) transport in biodegrading bacteria. The MAH channels show substantial structural differences compared to EcFadL, suggesting that MAH uptake occurs via a different mechanism compared to LCFA uptake. Moreover, preliminary data show that FadL channels are substrate specific. The current proposal will build on our work on EcFadL and other OM channels by determining how various AH are transported across the OM by the three FadL paralogs of PpF1. We will also assess which structural features underlie the substrate specificities of those FadL channels. Finally, we will determine the importance of FadL channel-mediated OM uptake of AH under conditions that mimic closely the natural environment. More specifically, we will pursue the following Aims: 1. To elucidate the transport mechanism of mono-aromatic hydrocarbons (MAH) across the OM. 2. To determine the structural basis for the substrate specificity of FadL channels. 3. To assess the importance of FadL channels for biodegradation. 4. To determine which proteins are important during toluene biodegradation by PpF1. To answer these questions we will combine a wide range of experimental approaches, including genetics, biochemistry, structural biology and microcosm experiments. The experiments will increase our knowledge about how hydrophobic molecules are transported across the OM and how substrate specificity is generated within channels that transport hydrophobic substrates. The results could potentially be used to design more efficient biodegrader and biocatalyst strains that utilize hydrophobic substrates.